Method for analysing a polynucleotide

ABSTRACT

A method of analysing a polynucleotide target involves incubating the target with an oligonucleotide probe, generally an array of immobilised oligonucleotide probes, to form a duplex, and using ligase or polymerase to extend one chain of the duplex. A point mutation or variable number tandem repeat section may be analysed. Arrays of immobilised oligonucleotides are provided for use in the method.

INTRODUCTION

Detection of variation in DNA sequences forms the basis of manyapplications in modern genetic analysis: it is used in linkage analysisto track disease genes in human pedigrees or economically importanttraits in animal and plant breeding programmes; it forms the basis offingerprinting methods used in forensic and paternity testing [Krawczakand Schmidtke, 1994]; it is used to discover mutations in biologicallyand clinically important genes [Cooper and Krawczak, 1989]. Theimportance of DNA polymorphism is underlined by the large number ofmethods that have been developed to detect and measure it [Cotton,1993]. Most of these methods depend on one of two analytical procedures,gel electrophoresis or molecular reassociation, to detect sequencevariation. Each of these powerful procedures has its drawbacks. Gelelectrophoresis has very high resolving power, and is especially usefulfor the detection of variation in the mini- and microsatellite markersthat are used in linkage analysis and fingerprinting; it is also themethod used to analyse the variation found in the triplet repeats thatcause a number of mutations now known to be the cause of around tengenetic disorders in humans [Willems, 1994]. Despite its great successand widespread use, gel electrophoresis has proved difficult toautomate: even the systems which automate data collection require manualgel preparation; and as samples are loaded by hand, it is easy toconfuse samples. The continuous reading electrophoresis machines areexpensive, and manual analysis is technically demanding, so that its useis confined to specialised laboratories which have a high throughput.Furthermore, difficulties in measuring fragment size preclude rigorousstatistical analysis of the results.

By contrast, oligonucleotide hybridisation lends itself to automationand to quantitative analysis [Southern et al., 1992], but it is not wellsuited to the analysis of variation in the number of repeats in themicro- and minisatellites, as the small fractional change in the numberof repeats produces a barely detectable change in signal strength; andof course it would not be possible to distinguish two alleles in thesame sample as each would contribute to a single intensity measurement.Thus, many different combinations of alleles would produce the samesignal. Present hybridisation methods are much better suited toanalysing variation in the DNA due to point mutation—base substitutiondeletions and insertions, for which it is possible to design allelespecific oligonucleotides (ASOs) that recognise both the wild type andthe mutant sequences [Conner et al., 1983]. Thus it is possible inprinciple, in a relatively simple test, to detect all possiblegenotypes. However, a problem that arises in practice in the use ofoligonucleotide hybridisation is that in some cases the extent ofreassociation is barely affected by a mismatched base pair.

THE INVENTION

The invention describes a general approach which can be applied to allforms of variation commonly used as DNA markers for genetic analysis. Itcombines sequence-specific hybridisation to oligonucleotides, which inthe preferred embodiment are tethered to a solid support, with enzymaticreactions which enhance the discrimination between matching andnon-matching duplexes, and at the same time provide a way of attaching alabel to indicate when or which reaction has taken place. Two enzymaticreactions, chain extension by DNA dependent DNA polymerases and DNAstrand-joining by DNA ligases, are dependent on perfect matching ofsequences at or around the point of extension or joining. As we shallshow, there are several ways in which these enzymes can be used withsequence-specific oligonucleotides to detect variation in targetsequences.

In all cases, the sequence to be analysed, the target sequence, will beavailable as a nucleic acid molecule, and may be a DNA moleculeproduced, for example, by the polymerase chain reaction. However, themethods are not confined to analysis of DNA produced in this way. In allapplications, the target sequence is first captured by hybridisation tooligonucleotides which are preferably tethered to a solid support; forexample, the oligonucleotides may be synthesised in situ as described[Maskos and Southern, 1992]; or they may be presynthesised and thencoupled to the surface [Khrapko et al, 1991].

In one aspect of the invention the novelty arises from the exploitationof enzymes in combination with substrates or primers tethered to solidsupports. A further novelty exploits the observation that DNA ligasesand polymerases can be used to distinguish sequence variants whichdiffer in the number of units of a tandemly repeating sequence. Thisobservation is surprising, as tandemly repeated sequences can formduplex in any register, thus in principle, length variants can formduplexes which match at the ends even when the two strands containdifferent numbers of repeat units. Although we demonstrate theapplication of this method in conjunction with tetheredoligonucleotides, it should be evident that this reaction could be usedto analyse VNTR (variable number tandem repeat) sequences in the liquidphase followed by some other method of analysis, such as gelelectrophoresis.

In one aspect the invention provides a method of analysis whichcomprises: providing a polynucleotide target including a nucleotide at aspecified position, and an oligonucleotide probe, tethered to a support,said probe being complementary to the target and terminating at or closeto the said specified position; and performing the steps:

-   -   a) incubating the target with the probe to form a duplex,    -   b) incubating the duplex under ligation conditions with a        labelled oligonucleotide complementary to the target,    -   c) and monitoring ligation in b) as an indication of a point        mutation at the specified position in the target.

In another aspect the invention provides a method of analysis whichcomprises: providing a polynucleotide target having a variable numbertandem repeat section and a flanking section, and an oligonucleotideprobe having a section complementary to the repeat section and aflanking section of the target; and performing the steps:

-   -   a) incubating the target with the probe to form a duplex,    -   b) incubating the duplex with a labelled oligonucleotide and/or        at least one labelled nucleotide under chain extension        conditions,    -   c) and monitoring chain extension as an indication of the length        of the variable number repeat section of the target.

A polynucleotide target is provided, in solution when the probe istethered to a support, and may be DNA or RNA. This polynucleotide targetis caused to hybridise with an oligonucleotide probe. The termoligonucleotide is used here, as common terminology far the primers andsubstrates commonly utilised by polymerase and ligase enzymes. However,the term is used in a broad sense to cover any substance that serves asa substrate for the enzymes, including single stranded chains of shortor moderate length composed of the residues of nucleotides or ofnucleotide analogues, and also longer chains that would ordinarily bereferred to as polynucleotides.

The probe may be tethered to a support, preferably by a covalent linkageand preferably through a 5′ or 3′ terminal nucleotide residue. An arrayof oligonucleotide probes may be tethered at spaced locations, forexample on a derivatised glass surface or the surface of a siliconmicrochip, or alternatively on individual beads.

In another aspect the invention provides an array of oligonucleotides,for analysing a polynucleotide target containing a variable sequence, inwhich each component oligonucleotide i) comprises a sequencecomplementary to the target including an expected variant of the target,and ii) is tethered to a solid support in a chemical orientation whicha) permits duplex formation with the target, and b)permits chainextension only when the sequence of the oligonucleotide matches thevariable sequence of the target.

In another aspect the invention provides a set or array ofoligonucleotides, for analysing a polynucleotide target containing avariable number tandem repeat sequence, in which each componentoligonucleotide i) comprises a sequence complementary to a part of thetarget immediately adjacent the repeat sequence, ii) comprises asequence complementary to the repeat sequence of the target andcontaining a number of repeats expected in the target, and iii) isconfigured in a way that a) permits duplex formation with the target,and b) permits chain extension only when the number of repeats in theoligonucleotide equals or is less than the number of repeats in thetarget.

In another aspect the invention provides an array of oligonucleotides inwhich different oligonucleotides occupy different locations and eacholigonucleotide has a 3′ nucleotide residue through which it iscovalently tethered to a support and a 5′ nucleotide residue which isphosphorylated.

The invention also provides a method of making an array of differentoligonucleotides tethered to different locations of a support, whichmethod comprises the steps of: providing a first intermediateoligonucleotide tethered to the support and a second intermediateoligonucleotide in solution, and a third oligonucleotide that iscomplementary to both the first and second intermediateoligonucleotides, forming a duplex of the third oligonucleotide with thefirst and second intermediate oligonucleotides, and ligating the firstintermediate oligonucleotide with the second intermediateoligonucleotide; and repeating the steps with oligonucleotides tetheredto different locations of the support

Reference is directed to the accompanying drawings in which each ofFIGS. 1 to 6 is a series of diagrams illustrating a method according tothe invention.

FIG. 1 shows detection of point mutation by single base extension.

FIG. 2 shows detection of point mutation by hybridisation to allelespecific oligonucleotides and chain extension.

FIG. 3A shows detection of point mutation by tag ligation to allelespecific oligonucleotides.

FIG. 3B shows detection of point mutation by ligation to library ofdifferentially tagged allele specific oligonucleotides.

FIG. 4A shows analysis of variable numbered tandem repeats by ligationof tag to allelic variants.

FIG. 4B shows analysis of variable number tandem repeats by ligation oftag to allelic variants.

FIG. 5 shows measurement of variable number tandem repeats by labelledchain extension.

FIG. 6 shows analysis of variable number tandem repeats by ligation oftag followed by chain extension.

DETAILED DESCRIPTION

Detection of Point Mutation

I. Single Base-Specific Extension of Tethered Primers.

In this application, the tethered oligonucleotide terminates at aposition one base before the variable base in the target sequence (FIG.1). A nucleotide precursor triphosphate or dideoxyribonucleotidetriphosphate, labelled, for example with a fluorescent tag, is added inthe presence of a nucleic acid synthesising enzyme which requires aspecific template in order to incorporate the complementary base. In thecase of DNA polymerase, the labelled base will be incorporated from adeoxyribonucleotide precursor only if the precursor base iscomplementary to the base in the target sequence. Thus, mutants willgive a negative result.

II. Chain Extension from Tethered ASOs.

In this case, the tethered oligonucleotide terminates in a base which iscomplementary to the variable base in the target sequence. Labelledprecursor nucleoside triphosphates and polymerase are added.Polymerisation takes place only if the last base of the primer iscomplementary to the variable base in the target (FIG. 2). Thus, mutantswill give a negative result.

III. Ligation of Tag Sequences to Tethered ASOs.

In this method, the tethered oligonucleotide may end at the variableposition in the target sequence, or it may end close to this position.In either case, hybridisation of the target to the tethered ASOs willproduce a substrate for ligating a tag oligonucleotide only if the basesat the join are well matched (FIG. 3 a). Thus, mutants which are closeenough to the joining position to prevent ligation will give a negativeresult. Alternatively, the tethered oligonucleotide may terminate at thebase before the variable position; in this case, the ligation reactioncan be carried out using a mixture of tag oligonucleotides, one for eachof the possible alternative variants. Each tag would be labelleddifferently, for example, with a different fluophore, so that those thatligated could be recognised identifying the variant base (FIG. 3 b).

Analysis of VNTR Lengths by Ligation to Anchored VNTRS

In this application “tag” oligonucleotides are ligated to sets oftethered oligonucleotides after hybridisation of the target, which actsas a template to bring the tags and the tethered oligonucleotidestogether.

In FIG. 4 a, the tethered oligonucleotides comprise three parts, an“anchor” sequence which is common to all members of a VNTR set, which isattached to the solid support and which hybridises to a sequenceflanking the variable region, a variable number of the repeated sequenceunit, and a distal sequence. Each allele, represented by a differentnumber of repeats, is located on a different solid support or at adifferent location on the same solid surface. Hybridisation of thetarget sequence will produce a series of duplexes, the structures ofwhich depend on the number of units that the target contains. If thenumber matches the number in a tethered oligonucleotide, the target willmeet the end of a tag when the tag is hybridised to the distal sequenceof the tethered oligonucleotide. If the number is greater or smaller,there will be a gap in the duplex which reduces or prevents ligation ofthe tag.

In FIG. 4 b, the tethered oligonucleotides comprise two parts: an“anchor” sequence which is common to all members of a VNTR set and whichhybridises to a sequence flanking the repeated region, and a variablenumber of the repeated sequence unit. Each allele, represented by adifferent number of repeats, is located on a different solid support, orat a different location on the same solid surface. Hybridisation of thetarget sequence will produce a series of duplexes, the structures ofwhich depend on the number of repeat units that the target contains(FIG. 4 b). If the number matches the number in a tetheredoligonucleotide, the latter will meet the end of the tag when the tag ishybridised to its complement in the target sequence, and form asubstrate for ligation of the tag. If the number is greater or smaller,there will be a gap in the duplex which reduces or prevents ligation ofthe tag.

Analysis of VNTR Lengths by Chain Extension

The number of repeat units in a VNTR may vary within small limits, andin these circumstances, the method of analysis described above, using aligase, will be appropriate; in other cases, for example thetrinucleotide repeats associated with a number of human inheriteddisorders, the variation may be too large to analyse in this way. For anumber of triplet repeats, the variation can be from around 10-50 in thenormal chromosome to more than a thousand in the affected individual(Table 1). It is probably unrealistic to measure such large numbers ofrepeats using the ligase reaction. In these cases, where the differencebetween the normal and the mutant allele is large, an alternative is tomeasure approximately the number of repeat units using labelledprecursors with a polymerising enzyme. The enzyme may be either apolymerase, such as DNA dependent DNA polymerase, or a ligase. In theformer case, the oligonucleotides have to be tethered at their 5′ endsto satisfy the requirement for enzymatic extension by the polymerase.The solid support carries an oligonucleotide anchor complementary to asequence flanking the repeat unit; for example, the sequence can be thatof one of the primers used to amplify the test sequence by the PCR.After hybridisation of the test sequence to the anchor, the repeatinsert may be copied by a polymerase or a ligase (FIG. 5) incorporatinga labelled precursor. The amount of label incorporated is proportionalto the number of repeat units. Incomplete hybridisation of the target tothe anchor sequence would give a deceptively low measure of the repeatnumber. This problem can be overcome by standardising the measurement inone of several possible ways. For example, if the target sequence itselfis labelled as shown in FIG. 5, the final measurement will be a ratio oftwo labels: the target and the incorporated precursor.

Alternatively, in the case of a triplet repeat, incorporation will endat the point in the sequence where the missing precursor base is neededfor further extension; where a ligase had been used to polymerisemonomers of the basic repeat unit, this will also end at the end of theVNTR insert. At this point a labelled “capping” sequence can be added byligation. In such cases, the measurement will be the ratio of cap topolymer labels.

Analysis of VNTR Lengths by Combining Ligation and Chain Extension

A more powerful way of analysing VNTRs which may vary in length over awide range, would be to test first for ligation to a labelled tagoligonucleotide; this would give the results already described fortargets with different lengths of repeats: a negative result where theVNTR lengths were longer or shorter in the target than in the tetheredoligonucleotides, and positive results where they were the same.Following ligation, which we have shown can be made to go to completion,the different length classes will behave differently as substrates forDNA polymerase. Those targets in which the repeat number is less thanthat of the tethered probe will not act as substrates (FIG. 6 c).Targets which have the same number of repeats as the probes will not beelongated by polymerase, because the ligated tag will block extension(FIG. 6 a). The only cases where extension will occur are those forwhich the targets are longer than the probes (FIG. 6 b). If the analysisis done on an array of probes with different numbers of inserts up to acertain limit, there will be a clear indication of the number of repeatsin the targets from the ligation results provided they are within therange of sizes represented in the array of probes. If, within thetargets, there is one which is longer than this range, it will show upin the polymerase analysis. This test will be especially useful for thetriplet repeats associated with the so-called “dynamic mutations”, forexample, that which is found in the fragile X mutation, where the sizerange varies from ca. 10-1000. It would be difficult to accommodate allof these size classes on a single array.

Experimental Support for the Claims

Properties of DNA Polymerases and Lipases

Most DNA polymerases, reverse transcriptases, some DNA dependent RNApolymerases and ligases can use as substrates one or moreoligonucleotides which are bound to a long DNA strand throughWatson-Crick base pairing. In the case of polymerases, anoligonucleotide is used as a primer to which the first base in thegrowing chain is added. In the case of ligases, two oligonucleotides arejoined provided that both are paired to the DNA strand and perfectlymatched in the base pairs at or close to the junction point. It is theseproperties that make the enzymes useful for the detection of DNAsequence variation; in particular, the requirement for specific basepairing at the site of extension or joining complements the sequencediscrimination that is already provided by the Watson-Crick pairingbetween the oligonucleotide and the target sequence that is needed toform a stable duplex. Thus, it has been found that discrimination byhybridisation alone is most sensitive if the variant base(s) is (are)close to the middle of the oligonucleotide. By contrast, for theenzymes, discrimination is highest if the variant mismatching bases areclose to the end where the extension or join takes place. Together,hybridisation under stringent conditions and enzymatic extension orjoining provide greater discrimination than either alone, and severalmethods have been developed to exploit this combination in systems forgenetic analysis [references in cotton, 1993]. The hybridisation and theenzyme reaction are normally carried out in solution, following whichthe product is captured on a solid support, or separated by gelelectrophoresis for detection and/or measurement.

In one embodiment, the invention described here employs oligonucleotidescoupled to a solid surface, so that the advantages of working in mixedphase are brought to all steps: hybridisation, enzymatic extension orjoining, and detection. This provides great sensitivity and convenience.As many different oligonucleotides can be bound to one surface in anarray, it enables many different sequences to be analysed together, in asingle reaction; this also ensures that all reactions are carried outunder identical conditions, making comparisons more reliable.

Support-Bound Oligonucleotides

Two different methods have been developed for making oligonucleotidesbound to a solid support: they can be synthesised in situ, orpresynthesised and attached to the support. In either case, it ispossible to use the support-bound oligonucleotides in a hybridisationreaction with oligonucleotides in the liquid phase to form duplexes; theexcess of oligonucleotide in solution can then be washed away.Hybridisation can be carried out under stringent conditions, so thatonly well-matched duplexes are stable. When enzymes are to be used, thechemical orientation of the oligonucleotide is important; polymerasesadd bases to the 3′ end of the chain; ligases join oligonucleotideswhich are phosphorylated at the 5′ end to those with a 3′-OH group.Oligonucleotides tethered to the solid substrate through either end canbe made in situ by using the appropriate phosphoramidite precursors[references in Beaucage and lyer, 1992]; or presynthesisedoligonucleotides can be fixed through appropriate groups at either end.We will demonstrate that oligonucleotides can be phosphorylated at the5′ end in situ using ATP and polynucleotide kinase, or they may bephosphorylated chemically [Horn and Urdea, 1986].

Tethered Oligonucleotides as Substrates for DNA Modifying Enzymes

The applications envisaged here require that the oligonucleotidestethered to the solid substrate can take part in reactions catalysed byDNA polymerases and ligases.

DNA Polymerase

The M13 sequencing primer—5′-GTAAAACGACGGCCAGT-3′—attached to aminatedpolypropylene through its 5′ end was synthesised as described. Asolution of M13 DNA (single-strand, replicative form, 0.1 μl, 200 ng/μl)was applied in two small spots to the surface of the derivatisedpolypropylene. A solution containing three non-radioactivedeoxyribonucleotide triphosphates, dATP, dGTP, TTP (10 μmol each),α²P-dCTP (10 μCi), Taq DNA polymerase and appropriate salts, was appliedover a large area of the polypropylene, including the area where the M13DNA had been spotted. The polypropylene was incubated at 37° C. for 1 hrin a vapour saturated chamber. It was then washed in 1% SDS at 100° C.for one minute, and exposed to a storage phosphor screen for one minuteand scanned in a phosphorimager. The regions where the DNA had beenapplied showed a high level of radioactivity, against a low backgroundwhere no DNA had been applied. This experiment shows thatoligonucleotides tethered to a solid support can act as primers forDNA-dependent synthesis by DNA polymerase, as required for applicationsusing this enzyme for mutation detection.

Experiments described below show that both polynucleotide kinase and DNAligase can be used to modify oligonucleotides tethered to a solidsupport. There are several ways in which phosphorylated oligonucleotidesand the ligase reaction can be used to detect sequence variation.

Methods for Making Arrays of Sequence Variants.

1. Allele Specific Oligonucleotides for Point Mutations.

For the preferred embodiment, it will be necessary to useoligonucleotides tethered to a solid support. The support may take theform of particles, for example, glass spheres, or magnetic beads. Inthis case the reactions could be carried out in tubes, or in the wellsof a microtitre plate. Methods for both synthesising oligonucleotidesand for attaching presynthesised oligonucleotides to these materials areknown (Stahl et al., 1988]. Methods for making arrays of ASO'srepresenting point mutations were described in patent applicationPCT/GB89/00460 and in Maskos and Southern (1993). We also demonstratedhow oligonucleotides tethered to a solid support in an array coulddistinguish mutant from wild type alleles by molecular hybridisation.

For the present invention, the same methods could be used to createoligonucleotide arrays of ASOs, but in order that they can be used assubstrates for the enzymes, they need to be modified; for ligation, itmay be necessary to phosphorylate the 5′ end; for extension bypolymerase it will be necessary to attach to oligonucleotides to thesolid substrate by their 5′ ends.

2. Arrays for Scanning Regions for Mutations.

It is often desirable to scan a relatively short region of a gene orgenome for point mutations: for example, many different sites aremutated in the CFTR gene to give rise to cystic fibrosis; similarly, thep53 tumour suppressor gene can be mutated at many sites. The largenumbers of oligonucleotides needed to examine all potential sites in thesequence can be made by efficient combinatorial methods [Southern etal., 1994]. A modification of the protocol could allow such arrays to beused in conjunction with enzymes to look for mutations at all sites inthe target sequence.

3. VNTRs.

The most commonly used VNTRs are repeats of very short units, typicallymono to tetranucleotides. However, there is another class, theminisatellites, in which the repeat unit is somewhat longer, up to 20 ormore nucleotides. The short repeats may be made using chemicalsynthesis; in the case of inserts with large numbers of repeat units, itwould be more economical to use a synthetic route which used the repeatunit as a reactant, rather than building them up one base at a time;such methods have been used to make polynucleotides by chemicalsynthesis. An attractive alternative would be to build the repeat unitsby ligating the monomer units; they could be added stepwise, one unit ata time provided a method could be found to block one end to preventpolymerisation; for example the oligonucleotide building block may beterminated by a hydroxyl group, which is then phosphorylated afterligation so that the unit becomes an acceptor for the next one; themonomer may have a phosphate group protected by a cleavable group, suchas a photocleavable group, which can be removed after ligation to allowa subsequent ligation [Pillai, 1980]. A second alternative, which wouldbe especially favourable for longer units such the minisatellites, wouldbe to attach either cloned or enzymatically amplified molecules to thesolid support. For example, each variant sequence could be amplified bythe PCR, using a biotinylated oligonucleotide for one of the primers.The strand starting with this group could then be attached to astreptavidin coated surface, and the other strand removed by melting[Stahl et al., 1988].

EXAMPLE 1

Demonstration of the Analysis of Length Polymorphism by Litgation to anArray of VNTRs

An array of VNTRs was made as described in FIG. 4 b, in which the anchorsequence was 5′-tgtagtggtgtgatcaaggc-3′. The repeat unit was 5′-cttt-3′;stripes, ca 3 mm wide, of sequence variants of the form:Anchor-Repeat_(N), with N=4-10, were made as stripes on the surface of asheet of polypropylene. The synthesis was carried out using3′-deoxyribophosphoramidites, this chemical orientation producesoligonucleotides tethered through their 3′ ends to the polypropylene,and a free 5′ hydroxyl group. To create a substrate for ligation, thisOH group was phosphorylated by immersing a strip of the polypropylene (3mm×18 mm), carrying the array of oligonucleotides, in 0.5 ml of asolution containing 4 mM ATP and 77.6 units of polynucleotide kinasewith buffer and Mg⁺⁺ according to the supplier's instructions. Thereaction was left for 6 hours at 37° C.; the strip was removed andimmersed in boiling water to kill the polynucleotide kinase. The targetsequences, which are complementary to elements of the arrayoligonucleotides and to the ligation tag, 5′-Anchor-Repeat₁₀-Tag and5′-Anchor-Repeat₅-Tag were added to 0.5 ml of a solution, preheated to95° C., containing the tag, 5′-gtggtcactaaagtttctgct-3′, which had beenlabelled at its 5′ end using polynucleotide kinase and ³³P-gamma-ATP,thermal ligase (500 units), and buffer and salts according to thesupplier's instructions. The polypropylene strip was immersed in the hotsolution, which was then allowed to cool to 68° C. , and left at thistemperature for 16 hours. The polypropylene strip was removed and placedin 25% formamide at 95° C. for 5 minutes, rinsed in water at the sametemperature, dried and exposed to a storage phosphor screen, from whichan image of the radioactivity was collected. The results showed countsclose to background over most of the array; counts on Anchor-Repeat₅ andAnchor-Repeat₁₀ were more than five times those over adjacent cells inthe array. This experiment indicates that the ligase is able todistinguish length variants of the repeat sequence and gives optimumligation only when the number of repeats in the target matches that inthe allele specific oligonucleotide in the array. Thus, it should easilybe possible to detect the two allelic variants in a heterozygote.

Ligation and/or polymerisation is possible if the oligonucleotide istethered through the 5′ end. The oligonucleotides can be synthesised insitu using deoxyribophosphoramidites with 3′ dimethoxytrityl groups and5′ phosphoramidite (reverse phosphoramidites). It is unnecessary tophosphorylate the tethered polynucleotide for ligation assays since thephosphate needed for ligation is provided by the tag oligonucleotide.

EXAMPLE 2

Analysis of VNTR Lengths

An array of VNTR's was made as described in FIG. 4B, with theoligonucleotides anchored through their 5′ ends. The repeat unit was 5′ttca and the anchoring sequence 5′ cttatttccctca. Stripes 6 mm wide ofsequence variants of the form: Anchor-Repeat_(N) where N=4-8 were madeon the surface of a sheet of polypropylene using “reverse”phosphoramidite monomers.

Analysis by Ligation

A strip of the array (30 mm×2 mm) was immersed in a solution of 600pmols of the target oligonucleotide 5′cacagactccatgg(tgaa)₆tgagggaaataag, 1.4 pmol of oligo 5′ ccatggagtctgtg(labelled at its 5′ end using polynucleotide kinase and ³³P gamma ATP)and buffer and salts according to the suppliers instructions, the totalvolume being 293 μl. The solution was heated to 65° C. and 7 μl of TthDNA ligase added. The reaction was then cooled to 37° C. and left atthat temperature for 18 hrs. After removal from the reaction solutionthe strip was washed in T.E. buffer, blotted dry and exposed to astorage phosphor screen from which an image of the radioactivity wastaken. The results showed that the target sequence ligated to thecorrect sequence with a higher yield than to the shorter and longersequences in adjacent cells of the array.

EXAMPLE 3

Analysis by Ligation and Polymerisation

A strip of the array (30 mm×2 mm) from Example 2 was added to a solutionof 200 pmols of the target oligonucleotide 5′cacagactccatgg(tgaa),tgagggaaataag, 200 pmol of oligo 5′ ccatggagtctgtg(chemically phosphorylated at the 5′ end) with buffer and saltsaccording to the suppliers instructions, the total volume being 243 μl.The solution was heated to 85° C. and cooled to 37° C. over a period of30 mins. 7 μl of Tth DNA ligase was added and the reaction mixtureheated at 34° C. for 17 hrs. The strip was removed and added to asolution of 8 mM DTT, 3.3 pmol ³²p alpha dTTP, 13 units sequenaseversion 2.0 and buffer and salts according to the suppliersinstructions. The total volume was 250 μl. After heating at 37° C. for 3hrs the strip was removed from the reaction solution, washed in TEbuffer, blotted dry and exposed to a storage phosphor screen from whichan image of the radioactivity was taken. The results showed counts equalto background over the area of the array where repeats were equal inlength or greater than the repeat length of the target, with 20 timesthe signal in the areas where the repeat length of the array was shorterthan the repeat length in the target.

EXAMPLE 4

Analysis by Polymerisation

Two types of polymerase analysis were carried out where reporternucleotides were chosen, in one case to identify the correct repeatlength, and in the other case to identify shorter repeat lengths. Thisis made possible when the repeat sequence comprises less than all fourbases.

In the former case a base is chosen which is present in the repeatsequence and is different from the first base in the flanking sequence.In the latter case a base is chosen to be complementary to the firstbase in the flanking sequence which is absent from the repeat.

A strip of the array from Example 2 (30 mm×2 mm) was added to a solutionof 500 pmols of the target oligonucleotide 5′cacagactccatgg(tgaa)₆tgagggaaataag in buffer and salts at aconcentration 1.09 times the suppliers instructions, the total volumebeing 275 μl. The solution was heated to 75° C. for 5 minutes and cooledto 37° C. over a period of 25 mins. The solution was removed and addedto 3.3 pmols ³²P alpha dCTP, 5 μl 1M DTT. 13 units of Sequenase version2.0 and water to give a final volume of 295 μl. This solution was addedto the array and heated at 37° C. for 15 hrs 40 mins. The polypropylenestrip was removed, washed in water and exposed to a storage phosphorscreen. The results showed counts of 5 times more for the correctsequence than shorter sequences and twice for the correct sequencecompared with longer repeats.

A similar strip of the array (30 mm×2 mm) was added to a solution of 500pmols of the target oligonucleotide 5′cacagactccatgg(tgaa)₆tgagggaaataag in buffer and salts 1.09 times thesuppliers instructions, the total volume being 275 μl. The solution washeated to 75° C. for 5 mins and cooled to 37° C. over a period of 25mins. The solution was removed and added to 3.3 pmols ³²P alpha dTTP, 5μl 1M DTT, 13 units of Sequenase version 2.0 and water to give a finalvolume of 295 μl. This solution was added to the array and heated at 37°C. for 15 hrs 40 mins. The polypropylene strip was removed, washed inwater and exposed to a storage phosphor screen. The results showedcounts of 4.5 times more for the shorter array sequences than thecorrect and longer repeat lengths.

EXAMPLE 5

VNTR Analysis by Ligation

In an experiment similar to the one described in Example 1, an array wascreated using the human fes/fps locus sequence as a target. The anchorsequence 5′ agagatgtagtctcattctttcgccaggctgg 3′ was the actual flankingsequence to the attt repeats of the fes/fps microsatellite (EMBLAccession No X06292 M14209 M14589) as it occurs in human genomic DNA.Using a target oligonucleotide representing the 10 repeat allele andligating a ³³P labelled 5′ flanking sequence (5′ g gag aca agg ata gcagtt c 3′) and doing a similar experiment to that described above, theresulting radioactivity on the anchor-repeat₁₀ cell was over 10 foldthat on adjacent cells in the array.

EXAMPLE 6

Demonstration of Stepwise Ligation of Oligonucleotides Bound to a SolidSupport.

A primer oligodeoxynucleotide—5′ PO₄ gta aaa cga cgg cca gt 3′, attachedto aminated polypropylene through its 3′ end, was synthesised andphosphorylated as described. A small square (2 mm×2 mm) piece of thismaterial was placed in standard ligation buffer, with templateoligonucleotide 5′tcg ttt tac cgt cat gcg tcc tct ctc 3′ (250 nM) and aprotected ligator oligonucleotide 5′ NB PO₄ cgc atg acg 3′ (250 nM) and33P labelled extender oligonucleotide 5′ gag aga gga 3′, where NB is aprotecting group based on a photocleavable o-nitrobenzyl derivative. TheNB protected phosphate of the ligator oligonucleotide had previouslybeen shown to be unable to take part in the ligation reaction. The NBgroup had also been shown to be removable by uv light to leave a fullyfunctional phosphate group. To this mixture was added thermusthermophilus DNA ligase (Advanced Biotechnologies) 25 u and the reactionincubated at room temperature for 6 hours. The mixture was thenirradiated with uv light (20 minutes room temperature) and incubated fora further 12 hours. The polypropylene patch was then washed with 30%formamide at 95° C. for 5 minutes, and exposed to a storage phosphorscreen for 24 hours and scanned in a phosphorimager. The patch showed alevel of radioactivity 50 fold higher than a patch treated in a similarfashion but without addition of the central “ligator” oligonucleotide.In a similar experiment using a phosphorylated ligator oligonucleotide asimilar amount of radioactive extender oligonucleotide became covalentlyattached to a third polypropylene/oligonucleotide primer square.

EXAMPLE 7

Demonstration of Point Mutation Analysis by Ligation to Allele SpecificOligonucleotides Attached to Solid Supports

Four tethered ASOs 5′ (gca or t)ag aga gga 3′, differing only at their5′ base, were synthesised as described above, with the 3′ end attachedto aminated polypropylene. Phosphorylation was carried out as describedand four squares of polypropylene carrying each ASO were placed instandard ligation buffer along with complementary target oligonucleotide5′ tcc tct ctc cgt cat gcg tat cgt tca at 3′ (250 nM). After addition of33P labelled ligator oligonucleotide 5′ cgc atg acg 3′ (10 nM) andthermus thermophilus DNA ligase (100 u), the mixture was incubated at37° C. for 18 hours. The ASO which was fully complementary to the targetoligonucleotide was found to have acquired 100-fold greaterradioactivity through ligation of the labelled ligator than thenon-complementary ASOs.

EXAMPLE 8

Demonstration of DNA Ligation Specificity

In a model experiment to assess the specificity of TTh DNA ligase,ligator and extender deoxyoligonucleotides were ligated together bymeans of hybridisation to an oligonucleotide template and ligation byDNA ligase.

Template oligonucleotide 5′ tcc tct ctc cgt cat gcg tat cgt tca at 3′(250 nM), phosphorylated, ³³P labelled, extender oligonucleotide 5′PO₄gag aga gga 3′(10 nM) and ligator sequence 5′ gca gta cg 3′ (250 nM)were mixed together in standard ligation buffer with DNA ligase 25 u.This mixture was incubated at 35° C. Samples of this mixture wereremoved and the reaction stopped by addition of formamide at 15, 30, 60,120, and 240 minutes. The ligated and unligated products were separatedby 20% denaturing polyacrylamide gel electrophoresis. The gel wasexposed to a phosphor screen for 18 hours and scanned by aphosphorimager. The relative proportions of ligated to unligatedproducts of the reaction were then measured. 50% of the extendersequence had been ligated to the ligator sequence in 30 minutes. Bycomparison in a similar experiment with ligator 5′ gca tga ag 3′ after30 minutes only 1% of the extender sequence had become ligated.

Other polymerases and ligases such as Taq polymerase, Thermosequenase,T4 DNA ligase and E. Coli DNA ligase have also been shown to be usefulin experiments similar to those described above.

REFERENCES

-   1. Beaucage, S. L. and lyer, R. P. (1992). Advances in the synthesis    of oligonucleotides by the phosphoramidite approach. Tetrahedron 48:    2223-2311.-   2. Conner, B. J., Reyes, A. A., Morin, C., Itakura, K., Teplitz, R.    L., and Wallace, R. B. (1983). Detection of sickle cell β⁵ globin    allele by hybridization with synthetic oligonucleotides. Proc. Natl.    Acad. Sci. USA 80: 278-282.-   3. Cooper, D. N., and Krawczak, M. (1989). The mutational spectrum    of single base-pair substitutions causing human genetic disease:    patterns and predictions. Hum. Genet. 85: 55-74.-   4. Cotton, R G, (1993) Current methods of mutation detection.    Mutation Research 285: 125-144.-   5. Horn, T., and Urdea, M. (1986) Chemical phosphorylation of    oligonucleotides. Tetrahedron Letters 27: 4705.-   6. Khrapko, K. R., Lysov, Yu. P., Khorlyn, A. A., Shick, V. V.,    Florentiev, V. L., and Mirzabekov. (1989). An oligonucleotide    hybridization approach to DNA sequencing. FEBS Lett. 256: 118-122.-   7. Krawczak M. and Schmidtke, J. (1994). DNA fingerprinting. BIOS    Scientific Publishers.-   8. Maskos, U., and Southern, E. M., (1993) A novel method for the    analysis of multiple sequence variants by hybridisation to    oligonucleotides. Nucleic Acids Research, 21: 2267-2268.-   9. Pillai, V. N. R.(1980). Photoremovable protecting groups in    organic chemistry Synthesis 39: 1-26.-   10. Southern, E. M. (1988). Analyzing Polynucleotide Sequences.    International Patent Application PCT/GB89/00460.-   11. Southern, E. M., Maskos, U. and Elder, J. K. (1992). Analysis of    Nucleic Acid Sequences by Hybridization to Arrays of    Oligonucleotides: Evaluation using Experimental Models. Genomics 12:    1008-1017.-   12. Southern, E. M., Case-Green, Elder, J. K. Johnson, M., Mir, K.    U., Wang, L., and Williams, J. C. (1994). Arrays of complementary    oligonucleotides for analysing the hybridisation behaviour of    nucleic acids. Nucleic Acids Res. 22:, 1368-1373.-   13. Stahl, S., Hultman, T., Olsson, A., Moks, T. D and,    Uhlen, M. (1988) Solid phase DNA sequencing using the biotin-avidin    system. Nucleic Acids Res. 16: 3025-38.-   14. Veerle, A. M. C. S., Moerkerk, P. T. M. M., Murtagh, J. J., Jr.,    Thunnissen, F. B. J. M (1994) A rapid reliable method for detection    of known point mutations: Point-EXACCT. Nucleic Acids Research 22:    4840-4841.-   15. Virnekas, B., Liring, G., Pluckthon, K., Schneider, C.,    Welihofer, G. and Moroney, S. E. (1994) Trinucleotide    phosphoramidites: ideal reagents for the synthesis of mixed    oligonucleotides for random mutagenesis. Nucleic Acids Research 22:    5600-5607.-   16. Willems, P. J. (1994) Dynamic mutations hit double figures.    Nature Genetics 8: 213-216.

DNA Sequences of Triplet Repeats Condition Repeat Normal PreexpansionExpanded Reference FRAXA CGG/CCG 10-50 38-50 200-1000  Verkerk et al.1991 FRAXE CGG/CCG 200-1000  Knight et al. 1993 FRAXF GCC/CGG  6-18 ?300-500  Parrish et al. 1994 FRAX16A CGG/CCG 1000-2000  Nancarrow et al.1994 SBMA CAG 11-31 ? 40-62  Tilley et al. 1994 Huntington CAG 11-3430-34 42-100  Huntington group 1993 SCA1 CAG 25-36 ? 43-81  Orr et al.1993 DRPLA/HRS CAG ≦100 Burke et al. 1994 Machado-Joseph CAG/CTG ˜26 ?68-79  Kawaguchi et al. 1994

1-13. (canceled)
 14. A method of analysis which comprises: providing apolynucleotide target including a nucleotide at a specified position,and two or more different oligonucleotide probes tethered to differentlocations of a support in the form of an array, each said probe beingcomplementary to the target including an expected variant of the targetand terminating at or close to the said specified position; andperforming the steps: (a) incubating the target with the probes to formduplex(es), (b) incubating the duplex(es) under (i) ligation conditionswith a labelled oligonucleotide complementary to the target or (ii)chain extension conditions with at least one labelled nucleotide; and(c) monitoring addition of the nucleotide/oligonucleotide to a probe byligation or chain extension in step (b) as an indication of a pointmutation at the specified position in the target.
 15. The method ofclaim 14, wherein an enzyme is used in step (b).
 16. The method of claim15, wherein the enzyme is a polymerase or a ligase.
 17. The method ofclaim 16, wherein the enzyme is a DNA polymerase, a reversetranscriptase, or a RNA polymerase.
 18. The method of claim 16, whereinthe enzyme is a Taq polymerase, a Thermosequenase, a T4 DNA ligase or aE. coli DNA ligase.
 19. The method of claim 14, wherein the probes aretethered by a covalent linkage.
 20. The method of claim 14, wherein theprobes are tethered through a 5′ or 3′ nucleotide.
 21. The method ofclaim 14, wherein the probes are tethered at spaced locations on aderivatised glass surface or on the surface of a silicon microchip or onpropylene.
 22. The method of claim 14, wherein the probes are tetheredto individual beads.
 23. The method of claim 14, wherein the labelledoligonucleotide or nucleotide is fluorescently labelled.
 24. The methodof claim 14, wherein labelled nucleoside triphosphates are added priorto step (c).
 25. The method of claim 24, wherein the nucleosidetriphosphates are deoxyribonucleotide triphosphates.
 26. A method ofanalysis which comprises: providing a polynucleotide target including anucleotide at a specified position, and an oligonucleotide probetethered to a support, wherein the probe is complementary to the targetand terminates one base before the said specified position; andperforming the steps: (a) incubating the target with the probe to form aduplex, (b) incubating the duplex under (i) ligation conditions with alabelled oligonucleotide complementary to the target and including anexpected variant of the target or (ii) chain extension conditions withone labelled nucleotide complementary to an expected variant of thetarget; and (c) monitoring addition of the nucleotide/oligonucleotide tothe probe by ligation or chain extension in step (b) as an indication ofa point mutation at the specified position in the target.
 27. The methodof claim 26, wherein an enzyme is used in step (b).
 28. The method ofclaim 27, wherein the enzyme is a polymerase or a ligase.
 29. The methodof claim 28, wherein the enzyme is a DNA polymerase, a reversetranscriptase, or a RNA polymerase.
 30. The method of claim 28, whereinthe enzyme is a Taq polymerase, a Thermosequenase, a T4 DNA ligase or aE. coli DNA ligase.
 31. The method of claim 26, wherein the probes aretethered by a covalent linkage.
 32. The method of claim 26, wherein theprobes are tethered through a 5′ or 3′ nucleotide.
 33. The method ofclaim 26, wherein the probes are tethered at spaced locations on aderivatised glass surface or on the surface of a silicon microchip or onpropylene.
 34. The method of claim 26, wherein the probes are tetheredto individual beads.
 35. The method of claim 26, wherein the labelledoligonucleotide or nucleotide is fluorescently labelled.
 36. The methodof claim 26, wherein labelled nucleoside triphosphates are added priorto step (c).
 37. The method of claim 36, wherein the nucleosidetriphosphates are deoxyribonucleotide triphosphates.
 38. A method ofanalysis which comprises: providing a polynucleotide target having avariable number tandem repeat section and a flanking section, and two ormore different oligonucleotide probes tethered to different locations ofa support in the form of an array, each said probe having a sectioncomplementary to the repeat section and a flanking section of thetarget; and performing the steps: (a) incubating the target with theprobes to form duplex(es), (b) incubating the duplex(es) under (i)ligation conditions with a labelled oligonucleotide complementary to thetarget and/or (ii) chain extension conditions with at least one labellednucleotide; and (c) monitoring addition of thenucleotide/oligonucleotide to a probe or target by ligation or chainextension in step (b) as an indication of the length of the variablenumber repeat section of the target.
 39. The method of claim 38, whereinthe polynucleotide target has a variable number tandem repeat sectionand two flanking sections, and wherein in step (b) labelledoligonucleotide(s) is/are ligated to the probe strand of the duplex(es).40. The method of claim 38, wherein an enzyme is used in step (b). 41.The method of claim 40, wherein the enzyme is a polymerase or a ligase.42. The method of claim 41, wherein the enzyme is a DNA polymerase, areverse transcriptase, or a RNA polymerase.
 43. The method of claim 40,wherein the enzyme is a Taq polymerase, a Thermosequenase, a T4 DNAligase or a E. Coli DNA ligase.
 44. The method of claim 38, wherein theprobes are tethered by a covalent linkage.
 45. The method of claim 38,wherein the probes are tethered through a 5′ or 3′ nucleotide.
 46. Themethod of claim 38, wherein the probes are tethered at spaced locationson a derivatised glass surface or on the surface of a silicon microchipor on propylene.
 47. The method of claim 38, wherein the probes aretethered to individual beads.
 48. The method of claim 38, wherein thelabelled oligonucleotide or nucleotide is fluorescently labelled. 49.The method of claim 38, wherein labelled nucleoside triphosphates areadded prior to step (c).
 50. The method of claim 49, wherein thenucleoside triphosphates are deoxyribonucleotide triphosphates.